Made available in DSpace on 2014-08-26T17:15:00Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 60.pdf: 1087420 bytes, checksum: d43c3b6da28a66b2ee5f2ae5c11fc7ae (MD5) Previous issue date: 2010 / Cronobacter spp. (Enterobacter sakazakii) são patógenos emergentes associados a surtos de meningite, enterocolite necrosante e septicemia em neonatos em diversos países. A taxa de mortalidade relatada varia de 40 a 80% e os sobreviventes apresentam seqüelas neurológicas severas. O desenvolvimento da infecção está associado ao consumo de fórmulas infantis desidratadas. As metodologias atualmente disponíveis para o isolamento de Cronobacter spp. apresentam discrepâncias e a identificação bioquímica não é esclarecedora. Assim, torna-se necessário que metodologias confiáveis para o isolamento e identificação desses micro-organismos estejam disponíveis. Outro ponto ainda não esclarecido são os mecanismos de virulência utilizados por esses patógenos para o desenvolvimento de infecções em humanos. Os objetivos desse estudo foram o desenvolvimento de um método rápido e eficiente para a identificação de Cronobacter spp., a avaliação do perfil de resistência a antimicrobianos e a avaliação da presença de alguns fatores de virulência. Foram utilizadas inicialmente cepas de referência de Cronobacter spp e 37 isolados previamente identificados como pertencentes ao gênero. O novo protocolo foi considerado 100% sensível e específico quando comparado aos demais métodos utilizados, indicando sua utlidade. O estudo de fatores de virulência revelou que Cronobacter spp. tem desenvolvido resistência aos β-lactâmicos e cefalosporinas, é capaz de formar cápsula, biofilme, e apresentar atividade de protease e atividade hemolítica. Nesse estudo relata-se pela primeira vez a presença de hemaglutininas associadas a pili tipo 3, enquanto algumas cepas apresentaram hemaglutininas associadas a pili tipo 1. Também foi demonstrada a atividade citotóxica em células Vero e a capacidade de invasão dessas células por Cronobacter spp. Os resultados desse estudo sugerem que todas as espécies do gênero Cronobacter apresentam potencial patogênico e que autoridades de saúde pública, produtores de alimentos e pessoas responsáveis pelo cuidado de indivíduos que fazem parte dos grupos de risco devem dedicar especial atenção ao controle de ambientes e à qualidade microbiológica de produtos destinados a esses grupos de modo a minimizar o risco de infecção por esses micro-organismos. / Cronobacter spp. (Enterobacter sakazakii) is an emerging foodborne pathogen associated with outbreaks of meningitis, necrotizing enterocolitis and sepsis in neonates in different countries. The reported mortality rate ranges from 40 to 80% and the survivors present severe neurological sequela. Development of the infection is associated with the consumption of powdered infant formula. The available methodologies employed for the isolation of Cronobacter spp. lack the necessary specificity and the biochemical identification remains non-conclusive. For these reasons, it is necessary to develop reliable methodologies for the isolation and identification of these microorganisms. Another issue still to be clarified refers to the virulence mechanisms associated with human infections. The objectives of this study were to develop a rapid and efficient method to identify Cronobacter, to evaluate the antimicrobial resistance of the strains, and to evaluate the presence of some virulence factors. Initially, reference strains of Cronobacter, and 37 isolates that had been previously identified as members of the genus were used. The proposed protocol showed 100% sensitivity and specificity when compared to the other methods used, indicating its usefulness. Studies on virulence factors revealed that Cronobacter spp. has developed resistance to β-lactams and cephalosporins, is able to form capsule, biofilm, and presents protease and hemolytic activities. This study reports, for the first time in literature, the presence of hemagglutinins associated with type 3 pili and it was also shown that some strains presented previously described hemagglutinins associated with type 1 pili. Cytotoxic activity in Vero cells and the invasiveness of these cells by Cronobacter spp were also demonstrated. The results of this study suggest that all species of the genus Cronobacter have pathogenic potential and public health authorities, food producers and caregivers should pay careful attention to issues associated with environment control and with the microbial quality of the products associated with the risk groups in order to minimize the risk of infection with these microorganisms.

Les produits alimentaires secs sont courants dans l’industrie agroalimentaire. Cependant, leur innocuité n’est pas toujours assurée et de nombreux cas de toxi-infections alimentaires collectives à travers le monde sont annuellement recensés. Deux bactéries pathogènes sont particulièrement impliquées, l’une correspond à grand nombre de cas, Salmonella enterica, et l’autre est reconnue pour sa capacité à résister aux perturbations environnementales, Cronobacter spp.. Une compréhension plus poussée de l’impact du séchage et traitement thermique à l’état sec peut permettre une optimisation des procédés de de décontamination des produits alimentaires secs et contribuer à réduire le nombre de toxiinfections alimentaires. Dans un premier chapitre, une attention particulière a été portée au procédé de séchage de microorganismes pathogènes qui peut, s’il est conduit de manière rapide, réduire considérablement la cultivabilité microbienne. De cette façon, le séchage peut être considéré comme une étape supplémentaire de décontamination à condition d’en optimiser les conditions d’application. Puis dans un deuxième chapitre, l’impact de la réhydratation, rarement traitée jusqu’à présent, a été étudié afin de proposer des conditions permettant de maximiser l’inactivation bactérienne. Cette étude remet partiellement en cause les dénombrements de bactéries pathogènes dans les aliments qui peuvent être, dans quelque cas, sous estimées du fait de l’utilisation de réhydratation rapide. Dans un troisième chapitre, l’application de différents traitements thermiques sur du lait en poudre, destiné à une population dite sensible, a permis de comprendre le rôle couplé de l’activité de l’eau et de la température sur l’efficacité du traitement thermique à l’état sec mais également de proposer un modèle d’inactivation thermique des pathogènes. Il a été notamment possible de définir un paramètre représentatif de l’effet de l’activité de l’eau (yaw). Les mécanismes de mort cellulaire pour les deux bactéries ont été ensuite étudiés, que ce soit après un séchage ou après un traitement thermique. Il apparait clairement que le séchage induit majoritairement une perméabilisation de la membrane tandis que le traitement thermique des cellules viables séchées dégrade majoritairement l’activité enzymatique en détruisant peu la membrane. Finalement, il a été montré que la virulence des deux pathogènes étudiés est augmentée par le séchage mais que le traitement thermique à l’état sec n’a pas d’impact sur cette virulence. Les connaissances apportées par cette étude peuvent être utiles d’un point de vue industriel, afin d’optimiser au mieux les conditions d’application des différents procédées pour une inactivation bactérienne maximale. / Dried food products are common in food industry. Nevertheless, their safety is not well insured, involving numerous outbreaks every years around the world. Particularly, two pathogenic bacteria are of interest, one of them due to its number of cases, Salmonella enterica, and the other one due to its ability to survive environmental perturbations, Cronobacter spp.. A deeper comprehension of drying and heat treatment in dried state impact could lead to an optimization of drying and heating processes, insuring food safety. In the first instance, drying was considered as a supplementary decontamination step by optimizing its conditions of use. In a second phase, the rehydration impact was studied in order to found optimal conditions of pathogen inactivation. The study questions the rehydration currently puts in practice for food safety analysis. In a third phase, several heat treatments on a dried food product, intended to susceptible population, permitted to understand the specific role of water activity on the efficiency of heat treatment in dried state, and also propose a modelling for bacterial heat inactivation. In addition, it was possible to define two new parameters which represent the effect of temperature (zT) and the effect of water activity (yaw). Fourth, cellular death mechanisms for both studied bacteria, during the drying or during the heat treatment, were considered. It was clear that drying involved mainly membrane damages whereas heat treatment involved mainly enzymatic damages. Finally, in a fifth part, it was shown that virulence properties of both studied bacteria were affected by drying, increasing invasion capacity, but not by heat treatment in dried state. To finish, the knowledges brought by this study are useful for industrial point of view, in the case of drying and heat process optimization and also by proposing biological indicators to valid process efficiency in situ.

Cronobacter sakazakii is a gram-negative opportunistic pathogen known to survive in dry environments and food matrices, such as infant formula. This foodborne bacterium can cause fatal human infections of the blood, central nervous system, and gastrointestinal tract; it is also problematic in wounds and urinary tract infections. Preterm infants and immunocompromised individuals are in higher risk categories related to necrotizing enterocolitis, neonatal sepsis, and meningitis due to this organism. Therefore, there is a need for increased understanding of how this bacterium is able to persist in thermally treated low-moisture products that do not support growth. The objective of this research is to identify genes and mechanisms in C. sakazakii that contribute to its resistance to desiccation and survival in low-moisture food matrices, including powdered infant formula. C. sakazakii sequence type 4 (ST4) is of particular interest as it is often the cause of neonatal infections originating from contaminated feedings of powder infant formula. The method chosen to explore these genetic patterns is massively parallel transposon insertion sequencing (Tn-seq). The E. coli strain MFDpir was used to facilitate transposon insertional mutagenesis to create a library of mutated C. sakazakii. Three different C. sakazakii ST4 isolates of different origins (clinical, environmental, and infant formula-derived) were selected for this study. Once transposon mutagenesis occurred with the aid of E. coli MFDpir, the three mutant libraries were subjected to desiccation stress in a closed system equilibrated to 11.3% relative humidity. The surviving mutant genomes were analyzed with Tn-seq. The sequencing data revealed that, while transposition events did occur successfully within the genomes of each of the selected C. sakazakii isolates, these events were not dense enough to draw biological conclusions nor statistical inferences concerning which genes contribute to this organism’s uncanny desiccation tolerance. However, we concluded that the Tn-seq method is a promising tool with this organism of interest, despite incomplete results in this first round of experimentation.

La presente tesis doctoral ahonda en dar respuesta a las exigencias de la FAO/OMS (2006) en cuanto al desarrollo de métodos bactericidas alternativos al térmico, aplicables a fórmulas infantiles reconstituidas (RPIFM) de preparados en polvo, principal vía de transmisión de un microorganismo patógeno emergente Cronobacter sakazakii. El principal objetivo sobre el que versan los estudios recogidos en la presente tesis doctoral es la aplicación de tecnologías no-térmicas de conservación, como barreras para controlar e inactivar el microorganismo C. sakazakii en RPIFM. La metodología de trabajo se basa en la aplicación de pulsos eléctricos de alta intensidad (PEAI) y altas presiones hidrostáticas (APH), mediante el uso de equipos a nivel de planta piloto. Se plantea también la adición de un ingrediente natural con capacidad antimicrobiana sobre la fórmula láctea tratada por PEAI. Además, se aplicarán herramientas de simulación para llevar a cabo una valoración de la exposición a C. sakazakii en ambiente hospitalario. Los principales resultados de la presente tesis doctoral ponen de manifiesto la efectividad de las tecnologías de PEAI y APH en el control del microorganismo alcanzando valores de inactivación que van desde [0.88-1.22] ciclos log10 a [2-7] ciclos log10 respectivamente. Ambas tecnologías dan lugar a la generación de células subletalmente dañadas, presentando éstas, una mayor sensibilidad frente a la aplicación de métodos barrera sucesivos, como en este caso, la refrigeración o la adición de un ingrediente natural con capacidad antimicrobiana. Por tanto, podemos concluir que la incorporación sucesiva de diferentes métodos de control, "tecnología de barreras", resulta eficaz para controlar e inactivar al microorganismo C. sakazakii en RPIFM. / Pina Pérez, MC. (2011). Aplicación de tecnologías no térmicas de conservación, pulsos eléctricos de alta intensidad (PEAI) y altas presiones hidrostáticas (APH), para el control de Cronobacter sakazakii en fórmulas lácteas infantiles: desarrollo de modelos predictivos y evaluación de la exposición [Tesis doctoral]. Editorial Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/14177 / Palancia